Deciphering of seed Health of common food grains (wheat, rice) of North Eastern UP and Gurgaon Haryana, India

The stored random samples of food seeds of wheat and rice (60 samples) were purchased from places of Eastern UP and Gurgaon district Haryana. Its moisture contents were estimated. The Mycological investigations of wheat seeds revealed presence of a total number of 16 fungal species viz., Alternaria alternata, Aspergillus candidus, Aspergillus flavus, A. niger, A. ochraceous, A. phoenicis, A. tamari, A. terreus, A. sydowi, Fusarium moniliforme, F. oxysporum F. solani, P. glabrum, Rhizopus nigricans, Trichoderma viride and Trichothecium roseum. While mycological analysis of rice seeds showed presence of 15 fungal species viz., Alternaria padwickii, A. oryzae, Curvularia lunata, Fusarium moniliforme, Aspergillus clavatus, A. flavus, A. niger, Cladosporium sp., Nigrospora oryzae, Alternaria tenuissima, Chaetomium globosum, F. solani, Microascus cirrosus, Helminthosporium oryzae, Pyricularia grisea. It also projected variation in presence of fungal species in blotter and agar plate method of analysis. In wheat Blotter method of analysis showed 16 fungal species while agar plate depicted 13 fungal species. In rice Agar plate method depicted presence of 15 fungal species while blotter method shows presence of 12 fungal species. The insect analysis revealed that wheat samples were infected with Tribolium castaneum. While rice seeds sample showed presence of insect Sitophilus oryzae. The investigations revealed that Aspergillus flavus, A. niger, Sitophilus oryzae and Tribolium castaneum caused reduction in seed weight loss, seed germination, carbohydrate and protein contents of common food grains (wheat, rice). It also revealed that randomly selected A. flavus isolate 1 of wheat showed higher potential of aflatoxin B1 production (1392.940 μg/l) while rice isolate 2 showed 1231.117 μg/l production.

India grows rice in 43 Mha with production of 112 million tons (Mt) of milled rice and average productivity of 2.6 t −1 ha 8 .
The food seeds (wheat, rice) are stored for varying length of time for various purposes. It is estimated that approximately 10-20% of the stored seeds become deteriorated by fungi. Several fungi have been associated with seeds viz., wheat seed 9,10 , rice seed 11,12 . The non scientific storage of wheat and rice seeds in rural areas of Eastern UP viz., Basti, Deoria, Gorakhpur, Maharajganj and Siddhartha Nagar and Gurgaon district Haryana viz., Farrukhnagar, Manesar, Pataudi, Sohna, Bilaspur leads to heavy deterioration by fungi and insects. However, detailed studies on such deterioration of stored seeds have not been made so far.
Keeping the above in view, in the present investigation, an extensive survey of stored seeds of wheat and rice were made for fungi and insects in order to find out its role in food seeds deterioration. Aflatoxin B1 production of Aspergillus flavus isolate were also investigated.

Materials and methods
Collection of stored seed samples of food. The random stored samples of food seeds (wheat, rice) [3 to 8 months old ]were purchased from places of Eastern UP viz., Basti, Deoria, Gorakhpur, Maharajganj and Siddhartha Nagar and Gurgaon district Haryana viz., Farrukhnagar, Manesar, Pataudi, Sohna, Bilaspur. From this all 10 selected spots from each spot six samples of food seeds (500 g) were purchased and kept separately in pre-sterilized polyethylene bags after labeling the name of district, tahsil and place. Thus all random 60 samples of seeds purchased were brought to Laboratory for analysis.
Estimation of Moisture content. Moisture content have role in seed mycoflora. So moisture content were estimated in all randomly collected 60 samples of food seeds (wheat, rice). The weight of 100 seeds (wheat, rice) were recorded randomly using a electronic balance. The seed moisture content was estimated following oven dry method using three replications each of 20 g (ISTA) 13,14 . After estimating the initial moisture content of seeds, about 200 g seed of each sample was kept in muslin cloth bags, to permit free flow of air, and placed in a seed drying room maintaining a constant temperature of 15 °C and 15% RH. Seed samples were drawn at an interval of seven days to estimate the moisture content. The per cent moisture content was estimated.
Detection of Seed Mycobiota. The mycofloral analysis of all randomly collected 60 samples of food seeds (wheat, rice) were done following techniques (i) Agar plate technique 15  The medium was sterilized in an autoclave at 20 lb/square inch pressure for 30 min. After cooling of medium to about 40 °C, 10 mg of Streptopenicillin was thoroughly mixed in them in order to prevent the bacterial contaminations 17 . 10 ml of medium was poured aseptically in each of the pre-sterilized petri plates (80 mm diam) separately and allowed to solidify. Glasswares used were pre-sterilized in an oven at 180 °C overnight. (ii) In standard blotter technique, three pieces of blotting paper were sterilized by dipping in ethyl alcohol. These were allowed to dry and placed inside a pre-sterilized Petri plate (80 mm diam).
Isolation of mycoflora from unsterilized seeds. 5 seeds of each sample of food seeds (wheat, rice) were kept equidistantly in each of the pre-sterilized Petri plates containing moistened blotters and solidified agar media separately.10 such assay plates were prepared comprising 50 seeds of each sample. Petri plates were incubated at 28 ± 2 °C for 7 days. The fungi appearing on the seeds were isolated, purified and their single spore cultures were maintained on Czapek's Dox agar medium in B.O.D. incubator at 10 ± 1 °C.
Isolation of mycoflora from sterilized seeds. All samples of food seeds (wheat, rice) were surface sterilized by dipping them 0.1%Sodium hypochlorite solution. The seeds were then washed thoroughly with sterilized distilled water to remove the traces of disinfectant. The seeds were placed on moistened blotters and solidified Czapek's Dox agar medium. The Petri plates were incubated at 28 ± 2 °C. The fungi appearing on the seeds were isolated on the seventh day.
The fungi were identified by comparing their morphological and cultural characteristics with authentic cultures maintained in Mycology Laboratory, Department of Botany, University of Gorakhpur and Amity Institute of Biotechnology, Amity University Haryana as well as with the help of available literature [18][19][20][21] .
The per cent frequency of unsterilized and sterilized seeds was calculated by using following formulae-Frequency (%) = No of plates in which individual fungal species occurred × 100/Total no. of plates studied.
Analysis of food seeds (wheat, rice) collected from different places for insects. Stored random samples (60) of food seeds (wheat, rice) [3-8 months old ] collected from places of Eastern UP viz., Basti, Deoria, Gorakhpur, Maharajganj and Siddhartha Nagar and Gurgaon district Haryana viz., Farrukhnagar, Manesar, Pataudi, Sohna, Bilaspur were also observed for their insect infestation. Observations for the occurrence of insects in samples of stored food grains were recorded as presence (+)/absence (−) of the insect in Table 4.
Deterioration caused by fungal species. Freshly harvested sterilized wheat, rice seeds were taken in pre-sterilized polyethylene bags (50 g/bag) and these were inoculated by one disc (5 mm diam) of different fungal species separately. For each fungal species 5 control and 5 treatment sets were made and stored for 20 days under laboratory conditions ( www.nature.com/scientificreports/ content of treated and control sets were observed. For germination seeds were placed on moistened filter paper and germination was recorded at different intervals for treated and control sets. The per cent germination was calculated by the following formula: Deterioration caused by insect species. The living insects viz., Tribolium castaneum, Sitophilus oryzae were collected in small glass tube (1′ × 4′) and plugged with cotton separately. 50 g surface sterilized healthy seeds of grains (wheat, rice) were placed in sterilized jars (in 5 replicate) with tin covers separately for each commodity.2 pairs of insects (2 males and 2 females) collected in glass tube were introduced in each jar having of one seed such as wheat, rice separately. Sterilized white hard paper strip was placed in jar for each movements of insects separately. A small pin size hole was made in each of the tin covers of the glass jar for gaseous exchange. The jars were placed in dark at room temperature (28 ± 2 °C). The observation in control and treatments were made after 2 months in terms of weight loss, germination percentage, Carbohydrate and protein content in each commodity inoculated seeds separately. The deterioration caused by fungi/insect in terms of carbohydrate content in wheat and rice seed were studied following Anthrone method 22 . The Carbohydrates were dehydrated through Conc. H2SO4 for forming furfural. Furfural then condenses with anthrone (10-Keto-9, 10 dihydro anthracene) to form a blue-green coloured complex. This was measured through calorimeter at 630 nm. The protein content estimation was done following Lowry et al. 23 by taking bovine serum albumin as standard. The optical density of each chickpea seed sample was taken at 650 nm.
Detection of aflatoxigenic isolates of Aspergillus species. Four isolates of A. flavus were randomly selected from each food seeds samples of wheat, rice separately to determine their Aflatoxin B 1 producing potential by Thin Layer Chromatography (TLC) 24 . Fifty μl conidial suspension (≈10 6 conidia/ml) of selected A. flavus isolates were separately inoculated in 49.5 ml SMKY (Sucrose, 200 g; MgSO 4 .7H 2 O, 0.5 g; KNO 3 , 0.3 g; Yeast extract, 7.0 g; Distilled water, 1000 ml) broth medium in 150 ml Erlenmeyer flask and mixed properly followed by incubation at 27 ± 2 °C for 10 days. Content of each flask was filtered after incubation and filtrate was extracted with chloroform (40 ml) in a separating funnel. The separated chloroform extract was dried on water bath at 60-70 °C. The residue left after evaporation was re-dissolved in 1 ml chloroform and 50 μl of it was spotted on TLC plate (20 × 20 cm 2 of silica gel). The plate was developed in toluene: isoamyl alcohol: methanol (90:32:2; v/v/v) solvent system and intensity of AFB 1 was observed under ultra violate fluorescence analysis cabinet at an excitation wavelength of 360 nm 25 . The fluorescent blue spots on TLC plate containing AFB 1 were scraped in 5 ml cold methanol and centrifuged at 3000 rpm for 5 min. Absorbance of supernatant was recorded at 360 nm and AFB 1 content was quantified 26 .

Results and discussion
It is evident from Table 1, that the 100 seed weight of wheat and rice seeds were in 4.60 ± 0.23, 2.68 ± 0.13(g) respectively which indicates the seed size diversity. After seven days of incubation grains showed 6.71 ± 0.53, 7.32 ± 0.43, per cent moisture content respectively.
A total number of 16 fungal species viz., Alternaria alternata, Aspergillus candidus, Aspergillus flavus, A. niger, A. ochraceous, A. phoenicis, A. tamari, A. terreus, A. sydowi, Fusarium moniliforme, F. oxysporum F. solani, P. glabrum, Rhizopus nigricans, Trichoderma viride and Trichothecium roseum were isolated by both agar plate as well as blotter paper methods from 60 random samples of places of Eastern UP viz., Basti, Deoria, Gorakhpur, Maharajganj and Siddhartha Nagar and Gurgaon district Haryana viz., Farrukhnagar, Manesar, Pataudi, Sohna, Bilaspur stored seeds of Wheat (Triticum aestivum L.) (Tables 2, 3 (Table 4). In wheat seeds the Blotter method of analysis showed 16 fungal species while agar plate depicted 13 fungal species (Table 4). Time to time researchers have isolated fungi the difference may be due to different climatic conditions. 25 genera and 59 species of seed-borne fungi from Egypt with the highest dominance of the genus Aspergillus (18 species + 2 varieties), followed by Penicillium (12 species + 1 variety, Fusarium on third in this regard (5 species + 1 variety), followed by Rhizopus spp., Mucor spp., Alternaria spp., and Curvularia spp. 27 ; A total of 28 genera and 72 species of seed-borne fungi, most common species viz., A. niger, A. flavus, A. terreus, A. nidulans, A. alternata, Cladosporium herbarum, and F. oxysporum 28 ; A. tenuis 29 ; Chaetomium globosum, Drechslera hawaiiensis, Fusarium subglutinens and Rhizoctonia solani by using blotter paper method 30 ; Fusarium spp., Bipolaris spp., Alternaria spp., Curvularia spp., Aspergillus spp., and Penicillium %Seed germination = No. of seed germinated × 100/Total no. of seed kept for germination It is interesting to note that sample of food seeds of Basti was badly infested from where maximum insect population was recorded (Table 6). Time to time previous investigators have reported observations on presence of storage insects on common seeds/grains. Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one of the major insect pests of stored seeds 51,52 . Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is the most important stored-product insect pest infesting rice (Oryza sativa L.) 53 .  www.nature.com/scientificreports/ As evident from Table 7, Aspergillus flavus, A. niger, Tribolium castaneum L. played important role in wheat seed weight loss and seed germination. The Aspergillus flavus inoculated wheat seeds showed 42.15 ± 0.19, A. niger 39.12 ± 0.14, Tribolium castaneum L 37.13 ± 0.16% Carbohydrate. content respectively. The Aspergillus flavus inoculated seeds showed 6.7 ± 0.14, A. niger 5.9 ± 0.15 while Tribolium castaneum L. inoculated wheat seeds showed 4.7 ± 0.19% protein content respectively (Table 7). Results reported that A. niger filtrate has a adverse effect on the germination rate of wheat seeds and the development of their seedlings. It could be due to the ability of the fungus to produce Aflatoxins. These findings are consistent with previous findings. Ijaz et al. 54 reported Table 5   www.nature.com/scientificreports/ A. niger as the most damageable storage fungi among fungal pathogens which leads to lower quality and seed germination. Culture filtrates of Aspergillus sp. have reported in causing a reduction in seed germination and root-shoot elongation 55 . The germination rate of wheat grains irrigated with the filtrate of A. niger and Rhizopus sp. was 20% and 80% respectively, compared with 100% of the control grains, which were irrigated with water. The culture filtrates of both A. niger and Rhizopus sp. affect not only percentage of grains germination but also the morphology of wheat seedlings 56 .
As evident from Table 8, Aspergillus flavus, A. niger, Sitophilus oryzae L. played important role in seed rice weight loss and seed germination. The Aspergillus flavus inoculated rice seeds showed 41.17 ± 0.11, A. niger 37.11 ± 0.12, Sitophilus oryzae 33.13 ± 0.13% Carbohydrate. content respectively. The Aspergillus flavus inoculated rice seeds showed 4.7 ± 0.11, A. niger 4.1 ± 0.10 while Sitophilus oryzae L. inoculated rice seeds showed 4.9 ± 0.10% protein content respectively (Table 8). It is evident from investigations that Aspergillus flavus, A. niger were dominant fungi causing harm to seeds of wheat and rice (Fig. 1). A study reported that there was high negative significant correlation between seed infestation by microflora and seed germination 57 . Jalander and Gachande 55 by study on the effect of different fungal species of seed-borne fungi of Aspergillus on germination and seedling growth of Bean and Cereals reported that A. niger caused reduction in germination percentage, growth of the plumule and radicle. As a negative impact, A. niger fungi affects on all rice seed germination characteristic more than all other fungi. In accordance with the results of the present study, Islam et al. 58 stated that there is negative and significant correlation [R = −97%] between rate of fungal contaminations and germination percentage in different rice cultivars. Among the studied factors, A. niger had high negative impact compared to other factors on all rice seed germination characteristics 59 .
As evident from Table 9, the randomly selected A. flavus isolate 1 of wheat showed higher potential of aflatoxin B 1 production (1392.940 μg/l) while rice isolate 2 showed 1231.117 μg/l production. While for both some isolates were non toxigenic. Other A. flavus isolates isolated from wheat and rice showed lower level of aflatoxin production.  www.nature.com/scientificreports/ Aflatoxins (AFs) are a group of mycotoxins produced as secondary metabolites by the spoilage of Aspergillus fungi, particularly Aspergillus flavus and Aspergillus parasiticus 60 . The most important members are aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ) and aflatoxin G 2 (AFG 2 ). They are highly toxic and carcinogenic compounds that cause disease in livestock and humans 61 . In recent years, numerous studies have revealed high levels of aflatoxins and fungal contamination in rice in many countries 62 . The maximum AFB1 concentration of 606 microg kg(−1) was observed in a wheat sample from the state of Uttar Pradesh 63 . AFB1 contamination in rice ranged from 0.014 to 0.123 µg/kg 64 . Out of 1200 rice samples, 67.8% showed AFB1 ranging from 0.1 to 308.0 microg/kg. All the paddy samples from Chattishgarh, Meghalaya and Tamil Nadu showed AFB1 contamination. Milled rice grains from different states showed below the permissible levels of AFB1 (average 0.5-3.5 micro g/ kg) 65 .

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.